EP0437281B1 - Composition comprenant un facteur de croissance de fibroblaste acide (aFGF) - Google Patents

Composition comprenant un facteur de croissance de fibroblaste acide (aFGF) Download PDF

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Publication number
EP0437281B1
EP0437281B1 EP91101195A EP91101195A EP0437281B1 EP 0437281 B1 EP0437281 B1 EP 0437281B1 EP 91101195 A EP91101195 A EP 91101195A EP 91101195 A EP91101195 A EP 91101195A EP 0437281 B1 EP0437281 B1 EP 0437281B1
Authority
EP
European Patent Office
Prior art keywords
afgf
growth
heparin
composition
endothelial cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP91101195A
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German (de)
English (en)
Other versions
EP0437281A1 (fr
Inventor
Kenneth A. Thomas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
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Merck and Co Inc
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Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0437281A1 publication Critical patent/EP0437281A1/fr
Application granted granted Critical
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Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)

Definitions

  • the subject invention relates to a composition for the stimulation of growth of vascular endothelial cells comprising aFGF and heparin.
  • Brain-derived acidic fibroblast growth factor, (aFGF) is an active mitogen for vascular endothelial cells in culture and is useful for growth of such cultures for coverage of polymeric vascular grafts; growth of such cultures on tubular supports for production of blood vessels for implantation; and stimulation or facilitation of blood vessel growth and repair in vivo .
  • the brain-derived aFGF purification and wound healing activity of this invention were described in US-A-4 444 760 issued April 24, 1984 to Kenneth A. Thomas, Jr.
  • the protein is also described by Thomas et al., Proc. Natl. Acad. Sci. , USA 81 , 357-361 (1984).
  • the same or similar proteins may also be present in other partially purified extracts from the central nervous system and other organs. See, for example, Maciag et al., Science , 225 , 932 (1984).
  • vascular endothelial cells Although the growth of vascular endothelial cells was accomplished in the past using very high concentrations of fetal calf or adult bovine serum (10-30%) the results were variable, depending on the particular lot of calf serum, and the rate of cell growth was generally slow. With aFGF rapid growth rates are achieved with serum levels from 0 to 2%.
  • the brain-derived aFGF used in the novel compositions of this invention is prepared as described in US-A-4 444 760.
  • the complete amino acid sequence has been determined for aFGF. Sequence determinations of reduced and carboxymethylated protein have revealed two amino termini. The longer sequence, aFGF-1, contains six amino terminal residues not found on the shorter aFGF-2 form. The relative amounts of these two microheterogeneous forms of the mitogen vary from one purification to another but are closely correlated in amount to the abundance of the two bands of protein previously seen by electrophoresis in SDS polyacrylamide gels (Thomas, et al., Proc. Natl. Acad. Sci. USA , 81 , 357 (1984)). As expected, the amount of the longer amino terminal sequence correlates with the relative quantity of the higher mass band on the SDS gels.
  • the length of the polypeptide chain at the amino termini is the only difference between the two microheterogeneous forms observed on the SDS gels, then the mass difference between them is 642 daltons, rather than the previously estimated 200 daltons based on SDS gel migration distances. It is assumed that the amino terminal heterogeneity is the result of limited proteolysis either in vivo or during purification.
  • the complete amino acid sequence was determined from sequences of the amino termini and overlapping peptides generated by proteolytic cleavages with trypsin (T), Staphylococcus aureus V8 Protease (V8), hydroxylamine (HA) and cyanogen bromide (CN).
  • T trypsin
  • V8 Protease V8
  • HA hydroxylamine
  • CN cyanogen bromide
  • aFGF is unique compared to the approximately 2000 protein sequences contained in that list.
  • compositions of the invention comprise heparin in addition to the aFGF discussed above. Those compositions can be used for neovascularization of surface wounds and for internal vascular growth.
  • the method for the stimulation of vascular endothelial cells comprises treating a sample of the desired vascular endothelial cells in a nutrient medium with aFGF at a concentration of about 1-10 ng/ml.
  • the concentration of heparin is about 10 to 100 ⁇ g/ml.
  • vascular endothelial cell growth is conducted in vitro , the presence of a nutrient medium such as Dulbecco's modified Eagle's medium or modification thereof is required and a low concentration of calf or bovine serum such as about 0 to 2 % by volume.
  • a nutrient medium such as Dulbecco's modified Eagle's medium or modification thereof is required and a low concentration of calf or bovine serum such as about 0 to 2 % by volume.
  • Preservatives such as a penicillin-streptomycin combination or other broad spectrum antibacterials are also employed.
  • the novel composition of this invention is useful for the coverage of artificial blood vessels with endothelial cells.
  • Vascular endothelial cells from the patient would be obtained by removal of a small segment of peripheral blood vessel or capillary-containing tissue and the desired cells would be grown in culture in the presence of aFGF and heparin and any other supplemental components that might be required such as serum. After growth of adequate numbers of endothelial cells in culture to cover the synthetic polymeric blood vessel the cells would be plated on the inside surface of the vessel. Prior coating of the artificial vessel either covalently or noncovalently, with either heparin or proteins such as fibrin, collagen, fibronectin or laminin would be performed to enhance attachment of the cells to the artificial vascular surface.
  • the cell-lined artificial vessel would then be surgically implanted into the patient and, being lined with the patients own cells, would be immunologically compatible.
  • the non-thrombogenic endothelial cell lining should decrease the incidence of clot formation on the surface of the artificial vessel and thereby decrease the tendency of vessel blockage or embolism elsewhere.
  • the novel composition is also useful for the production of artificial vessels.
  • Vascular endothelial cells and smooth muscle cells from the patient would be obtained and grown separately in culture.
  • the endothelial cells would be grown in the presence of the aFGF and heparin as outlined above.
  • the smooth muscle cells would be grown in culture by standard techniques.
  • a tubular mesh matrix of a biocompatible polymer either a synthetic polymer, with or without a coating of either heparin or specific attachment proteins, or a non-immunogenic biopolymeric material such as surgical suture thread
  • the novel composition can also be used for the induction of vascular growth.
  • the pure growth factor or the equivalent human protein and heparin would be used to induce and promote the growth of blood vessels in the patient.
  • the mitogen and heparin would be administered along with any necessary stabilizers and enhancers of activity at the site of desired vascular growth.
  • the formulation would be applied directly in a slow release polymer at a rate of about 1-100 ng/day/cm2 of injured surface.
  • the formulation would be released directly into the region to be neovascularized either from implanted slow release polymeric material or from slow release pumps.
  • the release rate in either case is preferably about 100 ng - 10 ug/day/cm3 of injured tissue.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Vascular Medicine (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Transplantation (AREA)
  • Organic Chemistry (AREA)
  • Neurosurgery (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Pulmonology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Ophthalmology & Optometry (AREA)
  • Dermatology (AREA)
  • Neurology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Claims (3)

  1. Composition pour la stimulation de la croissance de cellules endothéliales vasculaires comprenant de l'aFGF et de l'héparine.
  2. Composition de la revendication 1 pour la néovascularisation de blessures de surface.
  3. Composition de la revendication 1 pour la croissance vasculaire interne.
EP91101195A 1984-12-24 1985-12-16 Composition comprenant un facteur de croissance de fibroblaste acide (aFGF) Expired - Lifetime EP0437281B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US68592384A 1984-12-24 1984-12-24
US685923 1984-12-24
EP85116002A EP0186084B1 (fr) 1984-12-24 1985-12-16 Facteur de croissance dérivé du cerveau

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP85116002A Division EP0186084B1 (fr) 1984-12-24 1985-12-16 Facteur de croissance dérivé du cerveau
EP85116002.8 Division 1985-12-16

Publications (2)

Publication Number Publication Date
EP0437281A1 EP0437281A1 (fr) 1991-07-17
EP0437281B1 true EP0437281B1 (fr) 1994-04-13

Family

ID=24754216

Family Applications (2)

Application Number Title Priority Date Filing Date
EP91101195A Expired - Lifetime EP0437281B1 (fr) 1984-12-24 1985-12-16 Composition comprenant un facteur de croissance de fibroblaste acide (aFGF)
EP85116002A Expired - Lifetime EP0186084B1 (fr) 1984-12-24 1985-12-16 Facteur de croissance dérivé du cerveau

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP85116002A Expired - Lifetime EP0186084B1 (fr) 1984-12-24 1985-12-16 Facteur de croissance dérivé du cerveau

Country Status (4)

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EP (2) EP0437281B1 (fr)
JP (1) JP2510503B2 (fr)
CA (1) CA1307221C (fr)
DE (2) DE3587801T2 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4889808A (en) * 1985-10-01 1989-12-26 American Home Products Method of enchancing t-PA and SCU-PA production
US5827826A (en) * 1986-03-03 1998-10-27 Rhone-Poulenc Rorer Pharmaceuticals Inc. Compositions of human endothelial cell growth factor
US5552528A (en) 1986-03-03 1996-09-03 Rhone-Poulenc Rorer Pharmaceuticals Inc. Bovine b-endothelial cell growth factor
CA1322163C (fr) * 1986-07-03 1993-09-14 Kenneth A. Thomas, Jr. Production d'activateurs du plasminogene
NZ229354A (en) * 1988-07-01 1990-09-26 Becton Dickinson Co Treating polymer surfaces with a gas plasma and then applying a layer of endothelial cells to the surface
US5726152A (en) 1990-09-21 1998-03-10 Merck & Co., Inc. Vascular endothelial cell growth factor II
CA2051796A1 (fr) * 1990-09-21 1992-03-22 Marvin L. Bayne Facteur de croissance ii de l'endothelium vasculaire
US7109308B1 (en) 1994-03-08 2006-09-19 Human Genome Sciences, Inc. Antibodies to human vascular endothelial growth factor 2
US6608182B1 (en) 1994-03-08 2003-08-19 Human Genome Sciences, Inc. Human vascular endothelial growth factor 2
US6040157A (en) 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US7153827B1 (en) 1994-03-08 2006-12-26 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 and methods of use
US7186688B1 (en) 1994-03-08 2007-03-06 Human Genome Sciences, Inc. Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2
US5932540A (en) 1994-03-08 1999-08-03 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
JPH09510093A (ja) 1994-03-08 1997-10-14 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド 血管内皮細胞増殖因子２
US7223724B1 (en) 1999-02-08 2007-05-29 Human Genome Sciences, Inc. Use of vascular endothelial growth factor to treat photoreceptor cells
MXPA02003434A (es) 2000-08-04 2002-09-02 Human Genome Sciences Inc Factor de crecimiento endotelial vascular 2.
DK1385864T3 (da) 2001-04-13 2010-08-16 Human Genome Sciences Inc Anti-VEGF-2-antistoffer

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4444760A (en) * 1983-06-17 1984-04-24 Merck & Co., Inc. Purification and characterization of a protein fibroblast growth factor

Also Published As

Publication number Publication date
DE3587801T2 (de) 1994-10-20
EP0186084A3 (en) 1988-03-30
DE3587801D1 (de) 1994-05-19
CA1307221C (fr) 1992-09-08
JP2510503B2 (ja) 1996-06-26
EP0437281A1 (fr) 1991-07-17
DE3584005D1 (de) 1991-10-10
EP0186084B1 (fr) 1991-09-04
JPS61180720A (ja) 1986-08-13
EP0186084A2 (fr) 1986-07-02

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